Treating aids and HIV infection with methionine

ABSTRACT

This invention concerns novel safe compositions containing as an active antioxidant or antiinflammatory agent and thiol-repleting agent, the amino acid methionine, and/or one or more related compounds including certain metabolic precursor compounds, and novel methods employing the compositions for treating or amelio-rating symptoms of human immunodeficiency virus infection and AIDS, such as weight loss, increased red cell sedimentation, and leukoplakia; novel methods employing the compositions for treating or inhibiting symptoms resulting from nutritional deficiencies of methionine; and novel methods employing the compositions for inhibiting or preventing triglyceride elevation due to low dose methionine consumption. The compounds include the methionine hydroxy analogs, as well as compounds having the structural formula ##STR1## 1-, dl- or d- form and pharmaceutically acceptable N- (mono- and di-carboxylic acid) acyl derivatives and alkyl esters thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.07/479,961, filed Feb. 14, 1990 and now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the invention

This invention concerns improved antiinflammatory compositionscontaining the amino acid methionine (also known as "Met"), and/or oneor more related compounds including certain metabolic precursorcompounds with the amino acids glycine and serine to reduce side effectsof methionine at low doses and novel methods employing the compositionsfor ireating inflammatory AIDS symptoms and HIV infection in man.Compositions are suitable for the treatment of disease conditions of manthat may be attributable to or result from nutritional deficiency orillness resulting from uncontrolled inflammatory responses.

2. Description of the Related Art

A variety of efforts have been made over many years to elucidate themechanisms and origins of inflammation and the various forms of diseaseit may cause and to identify disease conditions which involveinflammatory damage. Limited success has been achieved in identifyingsuch conditions in alleviating the symptoms of diseases havinginflammatory components. Oxidative stress has been implicated in many ofthese diseases, and antioxidant therapy has been recommended as onemethod to alleviate the damage it causes (Cross, et. al., Annals ofInternal Medicine, 107:526-45, 1987). Successful prevention of oxidativestress damage requires that normal physiological parameters andfunctions be maintained so that undesired side effects do not occur.

It is now generally accepted that an essential component for theoccurrence of the disease known as AIDS (Acquired ImmunodeficiencySyndrome) is an infection by a virus now designated HumanImmunodeficiency Virus (HIV). This virus can be transmitted by theexchange of body fluids, including unprotected sexual intercourse, bloodtransfusions, and through sharing of non-sterile hypodermic needles.Blood borne occupational exposure is also known.

What has puzzled doctors and researchers in trying to understand thedisease of AIDS is the aspect of latency. Latency is the term used todescribe the long time period between a cause and its effect. Withregard to the progression of the AIDS disease from an early symptomlessstage of infection which can be demonstrated by the production ofantibodies to the virus to the eventual occurrence of symptoms typicalof the disease state, such as recurrent infections, weight loss,Karposi's sarcoma, etc., the period of latency can be longer than 7years. In one study (Arendrup, et al., Scand. J. Infect. Dis. 21:19-26,1989) 5 of 10 persons with HIV antibodies remained healthy over a 5 yearperiod. In most disease conditions the symptoms of the infection occurimmediately or soon after the infection. The progression from the stateof being healthy but showing evidence of infection with HIV to becomingsick with AIDS symptoms is associated with decrease in the number ofT-helper cells designated CD4, a decrease in the body's production ofantibody to the viral antigen p24, and increase in HIV antigen in theblood (Bottiger, et al., Scand J. Infect. Dis. 21:507-14,1989).

Because of the long latency that particular individuals show after HIVinfection it seems clear that other factors in addition the virus itselfcontribute to the occurrence of the disease symptoms. By understandingthe important elements operating that may allow infected individuals todelay the deleterious effects of the virus infection, considerablehealth benefits can be achieved for the antibody-positive, HIV infectedperson.

A benefit of compounds which modulate the activity of the immune systemof HIV-infected people is based on the hypothesis that the initialstages of the disease result in generalized immune system activation.Over time, this generalized stimulation results in activated immunecells which do not respond to further stimulation. The hypothesisincludes the concept of immune system failure after an extended periodof hyper-activity (Ascher and Sheppard, J. Acquired Immune DeficiencySyndromes, 3:177-91, 1990). The authors postulate that one possible modeof therapy is ". . . to target specific mediators that produce fever,cachexia, and diarrhea, and inhibit their action."

Recent identification of homology between the HIV viral proteindesignated "Nef" and a region of the histocompatibility antigens of HLAII, lends support to the hypothesis of an important involvement ofautoimmunity in leading to eventual HIV immunosuppression (Vega, M. A.,Guigo, R., and Smith, T. R., Nature 345: 26 (1990). The underlyingtheory is that the "Nef" protein region serves as a false signal to theimmune system mimicking that signal responsible for the stimulationwhich results when the histocompatibility antigen sites ofimmunoglobulins stimulate these cells as antigen-antibody complexes.

Severe liver damage occurs after consumption of large doses ofacetaminophen. Acetaminophen is converted by a cytochrome P-450 oxidaseto a reactive metabolism that is then inactivated by conjugation withglutathione. When large doses of acetaminophen are consumed, hepaticglutathione stores are depleted, and excess reactive metabolites causecell damage (Vale, et.al., Arch. Internal Med., 141:394-6, 1981). Thus,acetaminophen poisoning is a good model to study the effects of thioldepletion. N-acetylcysteine (NAC) and 1-methionine are the currentlyused antidotes for acetaminophen overdoses. Intravenous administrationof NAC can cause anaphylactoid reactions, and large oral doses ofmethionine can cause nausea and vomiting (Neuvonen, et al., Internat.Jour. Clinical Pharmacology, Therapy and Toxicology, 23:497-500, 1985).The addition of selenium and serine to the methionine and vitamin B6dosage allows the hepatic cells to maximize the production ofintracellular glutathione and extracellular glutathione peroxidase (Luo,et. al., Am.J. Clin. Nutrition, 42:4-48, 1985, and Sunde and Evenson, J.Biol. Chemistry, 262:933-7, 1987). The ability to maximize elutathioneand elutathione peroxidase production, while having an immediateantioxidant benefit and glutathione-sparing effect may be of value inother disease states where reduction of glutathione levels may be atoxic mechanism. Reduced glutathione effects include:

1) ataxia-telangiectasis (Meredith and Dodson, Cancer Research,47:4576-81, 1987);

2) human immunodeficiency virus infection (HIV) where blood levels ofglutathione are reduced in HIV positive persons (Buhl, et. al., TheLancet, Vol. 1 No. 8675, p. 1294-8, 1989);

3) rheumatoid arthritis (Braven et al., Br. J. Rheumatology, 28;212-15,1989; and Banford, et al., Rheumatology Internat 2:107-11, 1982);

4) Parkinson's Disease (Riederer, et. al., Jour. of Neurochemistry,52:515-20, 1989).

dl-Methionine is a chemically manufactured source of the essential aminoacid 1-methionine which contains an equal amount of the mirror imaged-methionine analog. The d-analog is not converted to the natural1-methionine by humans (although it can be utilized by animals such asdogs, chickens, etc.) and thus does not have nutritional value forprotein production. 1-Methionine is converted in the body tissues to theamino acid cysteine, the other sulfur amino acid present in mostproteins, by enzymatic reaction with another amino acid serine. Multiplebenefits of dl-methionine when taken with the amino acid serine, or theamino acid glycine, which is converted to serine in the body, arethought to arise from both the metabolic and anti-oxidant properties ofdl-methionine. The metabolic properties of 1-methionine include:

1. Providing methyl groups for many methylation reactions.

2. Providing homocysteine for the transulfuration pathway throughcystathionine for the production of the amino acid cysteine, which ispresent in high concentrations in hair.

3. Through cysteine production it provides glutathione, a three aminoacid compound which acts as an antioxidant in cells and in the blood.

4. Through cysteine production and the conversion of cysteine by thebody to hypotaurine, another antioxidant which is normally present inthe seminal fluid.

5. Through hypotaurine production and conversion in the body it providestaurine, another antioxidant amino acid, which is the highest inconcentration of all the free amino acids in cells. It is especiallyhigh in concentration in nerve cells and some white cells.

Additional serum antioxidant properties are provided by the use ofd-methionine which is due to the ability of d-methionine to providesustained elevated blood levels of methionine. This occurs becaused-methionine is poorly metabolized. However d-methionine is equally aseffective as 1-methionine as an anti oxidant. (Stegink, et al., Jour.Nutrition, 116:1185-92, 1986) and (Tsan and Chen, J. ClinicalInvestigation 65:1041-50, 1980).

The amino acid glycine may be included when seeking optimal benefits ofthe antioxidant methionine metabolites such as cysteine, glutathione,hypotaurine and taurine because when normal subjects are given 9 gramsof 1-methionine per day on an adequate diet, urinary glycine excretionlevels are reduced. No other urinary amino acid was altered (Block, etal., J. Nutrition, 86:256-64, 1965).

Consumption of oral methionine at 8 grams daily caused a 30% reductionin serum folate levels in five healthy subjects (Connor, et al.,Postgraduate Medical Jour., 54:318-20, 1978).

Excess methionine causes the urine to become acid and in so doingstimulates the excretion of calcium (Tschope, Mineral Electrolyte Metab.11:137-9, 1985). Calcium supplementation of about 20 milligrams per gramof methionine consumed would compensate this additional loss.

HIV-positive persons have benefitted from antioxidants. Studies withcompounds that act as water-soluble antioxidants often have seriousside-effects.

A recent study of 13 asymptomatic HIV infected patients treated withoral D-penicillamine demonstrated complete inhibition of virusexpression in 60% of those treated (Schulof, et al., Arzneim.-Forsch.Drug Res. 86 (II), No. 10, 1986). The basis for the human experiment wasa previous cell culture test which showed the D-penicillamine totallyinhibited viral growth at 40 microgram/ml, less that 10% of the cellulartoxic dose. Of 10 patients completing at least 2 weeks of treatment, 3tested negative for HIV six weeks after cessation of treatment bymeasurement of reverse transcriptase, antigen p15 and antigen p24. Sideeffects of the treatment protocol were significant with 4 of the 13patients developing skin rashes and 2 developing mild elevations ofhepatocellular enzymes (SGOT, SGPT) indicative of liver damage.

The rationale for testing D-penicillamine in HIV infection was based onits ability to interact with proteins and peptides by the formation ofmixed disulfides and thus inactivate cysteine-rich essential viralproteins, a high concentration of which are found in HIV.

D-penicillamine is used to treat rheumatoid arthritis where its benefitis thought to arise by reducing the damage done by the oxidativeproducts released during inflammation (Munthe. et al., In: Modulation ofAutoimmunity and Disease, Maini and Berry, Eds., Praeger, London, 1981,p. 134-42) and (Cross, et al., Annals of Internal Medicine, 107:526-45,1987). Of particular concern are the polymorphonuclear neutrophils(PMNs). These cells release relatively large amounts of oxidizers andproteolytic enzymes when activated (Weiss, New Eng. J. Medicine,320:365-76, 1989). They can cause wide-spread tissue damage, with oxygenmetabolites being identified as the most destructive toxins. Thus, inactive disease states due to infection, highly reactive free radicalscan be expected to be released by immune cells, including the PMN andthe macrophage.

The benefit of reducing compounds in the treatment of AIDS was shown ina double-blind clinical trial using diethyl-dithiocarbamate (DTC) as areducing agent. The treatment of HIV-positive persons once a week for a4 month period showed in the treatment group (1) fewer personsprogressing to AIDS, (2) a high percentage showing disappearance ofdiarrhea, weight loss or persistent fever, and (3) disappearance ofsplenomegaly. No change was detected in virus blood levels as measuredby reverse transcriptase activity (Lang, et. al., Lancet, Sep. 24, 1988,p. 702-6). In another study this same compound slowed the progression ofthe disease during the 6-month trial period (Reisinger, et. al., Lancet,335:679-82, 1990). DTC is known to interfere with glutathionemetabolism, while substituting for it as an anti-oxidant. When the AIDSpatients were removed from DTC, clinical benefits were progressivelylost.

In a separate study using DTC, which was administered by injectionrather than orally, there was significant improvement in symptoms and inreduction of lymphatic disease (Brewton, et al., Life Sciences 45:2509-2520, 1989). Benefits of DTC may be due to reduction of fungalinfections (Allerberger, F., et al., Mycoses 32: 527-530, 1989).

Free radical reducing compounds may benefit HIV patients as a result ofthe maintenance of blood and tissue glutathione levels. Glutathioneserves as an intracellular reducing agent and antioxidant (Deneke, etal.,. Am. J. Physiology, 257:163-73, 1989). Buhl, et al. (Lancet, Vol. 2No. 8675, Dec. 2, 1989) showed that symptom-free HIV positiveindividuals have only about half the level of plasma glutathione ascompared to healthy controls. A majority of HIV-positive patientstested, had abnormally low glutathione levels in lung epithelial liningfluid. Dworkin, et. al. (Biological Trace Element Research 15:167-77,1988) showed that glutathione peroxidase, which is an extracellularantioxidant, was 47% less in 13 AIDS patients, compared to 14 healthycontrols. Plasma selenium was also similarly reduced in AIDS patients.

In animal cell studies methionine is equally as effective as cysteine asa precursor for glutathione biosynthesis (Reed and Orrenius, Biochem,Hiophys, Res, Comm. 77: 1257-64, 1977). Rat feeding studies show thatmethionine can raise liver and muscle glutathione levels (Seligson andRotruck, J. Nutrition 113:98-104, 1983). In humans, the plasma half-lifeof a 100 mg i.v. dose of glutathione is 1.6 minutes (Wendel and Cikryt,FEBS Letters, 120: 209-11, 1980).

Kalebic, et al., (Proc Natl. Acad. Sci. USA, 88:986-90, 1991) show thatglutathione, glutathione ester, and N-acetylcysteine, at a dose of 15mM, were able to suppress HIV protein synthesis in vitro. The authorsspeculate that ". . . these or similar agents may have therapeutic valuein HIV-infected patients."

One mediator produced by the activated macrophage is a hormone called"cachectin" or "tumor necrosis factor". This hormone produces anorexiaand profound wasting then injected chronically into mice (Beutler, B.,Nutrition Reviews, 46: 369-73, 1988). It also causes the PMN(polymorphonuclear neutrophil white cell) to degranulate, release itsgranule contents, and activate the respiratory burst which releasesoxidative radicals (Balkwill, F. R., British Medical Bulletin, Vol. 45No. 2, p. 389-400, 1989 and Larrick, et al., Blood, 69:640-4, 1987).AIDS patients have been shown to have increased serum values ofcachectin/Tumor necrosis factor (Lahdevirta, et al.. Am. J. of Medicine,85:298-91, 1988). AIDS patients also show elevated levels of circulatingtriglycerides (Grunfeld, et al., Am. J. of Medicine 86:27--, 1989) whichmay be an effect of cachectin which has been shown to inhibitlipoprotein lipase (Fried and Zechner, J. of Lipid Research, 30:1917-23,1989).

Tumor necrosis factor stimulates the production of HIV virus in MOLT-4cells while another neutrophil (PMN) activator, phorbol 12-myristate13-acetate, stimulates HIV virus replication in human peripheral bloodmononuclear cells. N-acetyl cysteine, (NAC) an antioxidant utilized tocounteract oxidative stress such as that which occurs with acetaminophenoverdose, and which also increases intracellular glutathione, has beentested for its effect on virus replication in MOLT-4 cells andmononuclear cells (Roederer, M., et al., Proc. Natl. Acad. Sci. USA 87:4884-4888, 1990). NAC at 3 mM reduces virus production in MOLT-4 cellsby more than half, but more than 10 mM is required to reduce virusproduction by this amount when the virus production is stimulated bytumor necrosis factor. In human mononuclear cells, NAC reducesphorbol-induced virus production by more than half at 1 mM. More than 3mM NAO is required to reduce virus production in infected, unstimulatedmononuclear cells. Doses of NAC required to achieve blood or tissueslevel above 3 mM are much higher than those achieved by previouslyapproved uses of NAC.

PMNs have also been demonstrated to predominate in the cerebrospinalfluid (OSF) of AIDS patients who have cytomegalovirus infection in thenervous system (de Gans, J., et al., J. of AIDS, 3:1155-8, 1990).

Anemia is another complication of AIDS. Anemia of chronic disease iscommonly seen, and may be due to inflammatory mediators released bymacrophages. Controlling inflammation in AIDS may allow blood values tonormalize (Baer, et al., Seminars in Arthritis and Rheumatism, 19 (4):209-23, 1990).

A recent study in mice showed that infection with influenza virus led toa forty-fold increase in an enzyme called xanthine oxidase. This enzymecauses a generalized oxidative reaction in tissue. Administration ofsuperoxide dismutase, a free radical scavenger, could prevent death inmice which would otherwise occur. Virus titers dropped by day four,while xanthine oxidase conversion peaked on day eight, when deathoccurred, unless SOD was intravenously administered. The implication ofthese studies is that the oxidative attack generated by xanthine oxidasewas the direct cause of death, not the virus (Oda, et. al., Science,244:974-6, 1989). Xanthine oxidase is produced in tissues by theoxidation of exposed sulfhydryls in xanthine dehydrogenase. Thisoxidation can be accomplished by oxygen introduced during reperfusion,or presumably by oxidative products of PMN (Parks and Granger, ActaPhysiol. Scand. Suppl. 548:87-99, 1986). Another study, using thehamster cheek pouch method, shows that methionine was able to completelyinhibit the vascular changes which xanthine oxidase would otherwiseinduce (Del Maestro, et al., In: Biological and Clinical Aspects ofSuperoxide and Superoxide Dismutase, No. 62, Bannister and Bannister,Eds., New York, 1978, p. 127-40). This inhibition was believed to be dueto the known hydroxyl radical scavenging effect of methionine.Methionine has been shown to inhibit both PMN oxidative products as wellas xanthine oxidase free radical production. It has been hypothesizedthat the damage seen in AIDS is oxidative in nature, and that the use ofappropriate antioxidants is warranted (Papadopulos-Eleopulos, E.,Medical Hypotheses 25:151-62, 1988).

In addition to its potential antioxidant benefits, dl-methionineprovides 1-methionine for supplemental nutrition. Methionine and cystineblood levels are reduced 25% in AIDS patients (Droge, et al., Biol.Chem. Hoppe-Seyler 369:143-8, 1988), which further reduces the body'sability to produce glutathione (see above). This reduction issignificant in light of the fact that plasma methionine levels were notreduced in eight healthy subjects after seven days of starvation(Martensson, J., Metabolism 85:118-21, 1986).

Another problem found in pediatric AIDS is a large reduction of folicacid (80% of normal) in cerebral spinal fluid (OSF), which may cause thedemyelination of nerve fibers seen in AIDS. Accompanying the folatereduction was a 50% reduction of CSF S-adenosyl-methionine, theprincipal methyl donor in the brain (Surtees, et al., The Lancet,335:619-21, 1990).

Folate deficiency has also been reported in HIV infected adults atvarious degrees of progression of the disease. Simple folatesupplementation corrected this deficiency (Boudes, P., Zittoun, J., andSobel, A., Lancet 335: 1401-1402, 1990).

The pharmacopeia advises that methionine should not be taken by personswith active kidney or liver disease. Persons on chemotherapy takingfolate inhibitors such as methotrexate or trimetrexate should not takemethionine or folate.

Methionine is not recommended for persons being treated with monoamineoxidase inhibitors such as deprenyl. Schizophrenic patients given 300 mgper kg of 1-methionine per day exhibited symptoms of intoxication (TheMartindale Pharmacopoeia, 26th Ed., The Pharmaceutical Press, London, p76, 1972)

Methionine deficiency is not recognized as a disease state in moderncountries where adequate total protein is generally available. While itis recognized that humans, in contrast to most other mammals, cannotutilize d-methionine as a source of methionine, it is generally assumedthat humans can utilized methionine sulfoxide as a source of methionine.The only suggestion that methionine sulfoxide might not be nutritionallyequivalent to methionine is the lack of increase of blood 1-methionineafter administration of 1-methionine sulfoxide. Human enzymes have beenfound that can reduce methionine sulfoxide to methionine.

Methionine is known to be affected by a variety of food processingactivities. 1-Methionine is converted to d-Methionine when proteins areheated and a significant amount of the nutritional value of methioninecan be lost by this mechanism. However, most of the potential loss ofavailable methionine occurs through the mechanism of oxidation ofmethionine to methionine sulfoxide. The bleaching of flour may be themajor cause, when during the process of bleaching the chlorine is ableto react with methionine. When proteins are heated with reducing sugarsmethionine is readily oxidized so that items such as fruit "canned" innatural sugars are potential sources of food source with a deficiency ofavailable methionine. More recently, as unsaturated fats replacesaturated fats in prepared food sources, additional cases of methionineoxidation occur. For example, the unsaturated fats in cake-mixes held ina hot warehouse would result in oxidation of methionine to itssulfoxide. Published evidence for an extensive loss of methionine infood processing as regards human nutrition occurred in the manufactureof instant oatmeal where the product used in nitrogen balance studiesapparently had no nutritionally available methionine (see Kies, et. al.,J. Nutrition, 105:809-4, 1975). In the cooking of several types of beans40% to 50% of the methionine is not available to rats (Sawar and Peace,J. Nutrition. 116:1172-84, 1986). The dietary requirement for methionineplus cysteine is based on nitrogen balance studies where a total of 800mg per day is required to bring 50% of adults into positive nitrogenbalance. No attempt has been made to determine the level of methioninethat might be optimal for the prevention of oxidative damage.

Methionine has been shown to be a target for the products of stimulatedpolymorphonuclear neutrophils (Tsan and Ohen, J. Olin. Invest.,65:1041-50, 1980). The granular fraction of the PMNs oxidizes methionineto its sulfoxide in the presence of peroxide. Peroxide does not oxidizemethionine to its sulfoxide at normal physiological concentrations.

Some of the differences measured in the relative effectiveness ofmethionine compounds and other chemicals, especially sulfhydryl reducingsubstances, may be attributed to the control mechanisms that operate inanimals and man to regulate the amounts of these substances, wheregiving more of a substance does not significantly increase blood andtissue levels of that substance. Stegink, (Jour. Nutrition,116:1185-92,1986), showed that 0.5 gm of methionine elevated total blood methionine2-fold for 2 hours with 1-methionine but 3-fold for 4 hours withd-methionine. In the same study it was shown that methionine sulfoxideadministration did not result in elevation of blood methionine. Thisobservation suggests that methionine sulfoxide is not readily reduced tomethionine but it is possible that this reduction occurs in tissueswhere the methionine remains sequestered. 1-Methionine is an essentialamino acid for human nutrition. The normal serum level of methionine inman is 15 ppm. dl-Methionine is available as a one-a-day food supplementin 500 mg. oral tablet form.

Regarding human nutrition, 1-methionine is an essential amino acidwhereas d-methionine is non-nutritive. For purposes of metabolism,1-methionine via S-adenosylmethionine has an important methylatingfunction. In this function it loses a methyl group from its sulfur atomto become homocysteine. Homocysteine in excess can lead tohomocysteinuria, and may be heart disease associated (Malinow, et al.,Circulation 79:1180-88, 1989 and Olszewski and Szostak, Atherosclerosis69:109-13, 1988). Folic acid has been shown to be an innocuous method toreduce plasma homocysteine levels (Brattstrom et al., Scand. J. Clin.Lab. Invest. 48:215-221, 1988). Administration of 8 grams of1-methionine to adult subjects for four days caused a greater than 30%reduction in serum folate levels (Conner, et al., PostGrad. Med. J.54:318-20, 1978).

Administration of large amounts (5 to 10 grams per day) of 1-methioninecan cause gastrointestinal upset. Many people report a burning sensationin the stomach after taking methionine, along with an upset stomach andflatulence (Delrieu, et al, Revue du Rhumatisme, 55:995-7, 1988) Entericcoating and timed-release formulations should avoid the stomach problemsand allow even elevations of blood methionine for maximum anti-oxidanteffect. Typical enteric coating agents include cellulose acetatephthalate, and other cellulose ethers and derivatives (Johnson, J. C.,in Sustained Release Medications, Noyes Data Corp, N. J., 1980, p.14).

The Food and Nutrition Board of the U.S. National Academy of Scienceshas established the Recommended Daily Allowance (RDA) for nutrients formost healthy individuals. For a discussion, see The Nurses Guide to DrugTherapy, Eisenhauer and Gerald, Prentice-Hall, New Jersey, 1984-5, pages84-602). RDA's include: Vitamin A: 5000 I.U.; Vitamin B12:3 mcg.;Vitamin B6: 2 mg; Vitamin B3: 18 mg ; Folic Acid: 400 mcg.; Vitamin C:100 mg ; Vitamin D: 400 I.U ; Vitamin E: 15 I.U : Calcium: 800-1200 mg.;Iron: 18 mg.: Selenium: No RDA has been established, but the maximumnon-toxic suggested dose is 200 mcg. per day; zinc: 15 mg. For a reviewof lifestyle risk factors and of protective factors in the diet, see thearticle by Bruce N. Ames entitled "Dietary Carcinogens andAnticarcinogens", Science, 221:256-68, 1983.

Epidemiological evidence in man correlates elevated blood level oftriglycerides with increased occurrence of gallstones. Also, low serumcholesterol levels are associated with a high prevalence of gallstonedisease. Thus, dietary methods to lower serum cholesterol may increasethe risk of gallstones if serum triglyceride levels are not lowered atthe same time. (Angelico, et al., In: Epidemiology and Prevention ofGallstone Disease, Capocaccia, L., et al, eds. MTP Press Ltd., Boston,1984, p. 77-84). Yanagita et al., (Agricultural and BiologicalChemistry, 48:815-6, 1984) show that administration of methionine torats causes a doubling of hepatic triglycerides. However, Sugiyama, etal , (Agric. Biol. Chem., 49:3455-61, 1985) showed that the addition ofglycine to a methionine supplemented diet had no effect on the elevationof triglycerides caused by the methionine.

The patent to Scheinberg U.S. Pat. No. 4,315,028 describes a method oftreatment of arthritis employing substituted cysteines.

The S-methyl derivative of methionine, S-methyl methionine, also knownas vitamin U has been shown to have benefit as an anti-ulcer compoundand to have benefit for allergies. The same benefit is shown forcarboxyl esters and N-acyl derivatives (Kowa, DT 282-704). However, inthis teaching no distinction is made for the d- and 1- isomers ofS-methyl methionine or its derivatives and no claim is made that thesecompounds act through anti-inflammatory mechanisms.

The patent to Damico, U.S. Pat. No. 3,952,115 describes foodstuffscontaining N-acyl 1-methionine esters and N-acyl 1-cysteine esters.d-Isomers are specifically excluded because they are "not nutritionallyavailable".

The patent to Fahim, U.S. Pat. No. 4,711,780, shows the benefit of thecombination of cysteine with vitamin C and zinc salts in a topicalmixture for stimulating cell proliferation. The benefit of methionine isclaimed but not shown. No demonstration of benefit or claim for systemicadministration is made.

The patent to Bayless, U.S. Pat. No. 4,902,718, describes the use ofmethionine compounds for respiratory. The patent to Bayless, U.S. Pat.No. 4,927,850, describes the use of methionine compounds for calciumnormalization, hypertension and osteoporosis. The patent the Hirsch,U.S. Pat. No. 5,053,429 describes the use of methionine compounds forinflammatory pain. The patent to Hirsch, U.S. Pat. No. 5,084,482,describes the benefits of methionine compounds for ischemic thromboticand cholesterolemic diseases. These patents also teach the use ofglycine and serine alone or in addition with vitamins and minerals toreduce the side effects which are associated with elevated homocysteineresulting from methionine consumption.

From the extensive infection of humans with HIV and the impendingepidemic of AIDS there is an immediate need for improved and safe meansof treating disease conditions and delaying the onset of symptoms ofAIDS.

It is an object of the invention to provide methods for the treatment ofdisease conditions and delay the onset of symptoms which occur in AIDS.

It is also an object of the invention to provide means prevent oralleviate elevation of serum triglycerides that may result from excessmethionine intake.

It is also an object of the invention to provide a safe means of thiolrepletion.

These and other objects, features and advantages will be seen from thefollowing detailed description of the invention.

SUMMARY OF THE INVENTION

Our invention is based on the discovery that certain methionine ormethionine-type compounds in the dl-form at relatively high,well-tolerated doses are potent antioxidant and antiinflammatory agentsin man which are of benefit to persons infected with the HumanImmunodeficiency Virus (HIV). The compounds are especially importantwhen administered or used for treatment in dosage form, for relieving,inhibiting or abolishing any of various inflammatory disease conditionsor syndromes presenting as thiol depletion, which include HIV infection.The methionine compounds in high daily dosage according to the inventionthus may act in vivo to inhibit oxidative effects on sulfhydrals such asthe action of hypochlorous acid to reduce proteolysis and tissue damage.

Novel methods and compositions for the prevention and treatment ofdisease conditions of man may be attributable to or result fromnutritional deficiency of the 1-form of methionine, such as glutathionedepletion, are also disclosed.

Novel methods and compositions for the prevention of side-effects due tochronic administration of low doses of methionine in man by inclusion ofglycine or serine for the normalization of serum triglyceride levels aredisclosed.

For purposes of the invention, one uses at least one methionine-typecompound selected from the methionine hydroxy analogs and methioninecompounds having the structural formula I ##STR2## 1-, dl or d form andpharmaceutically acceptable N- (mono- and di- carboxylic acid) acylderivatives and alkyl esters thereof, where n is an integer from 1 to 3.

Thus, the methionine-type compound (for convenience some times referredto herein as "methionine" or "methionine compound") may be normethionine(n=1), methionine (n=2), homomethionine (n=3), the hydroxy analog, orthe acyl or alkyl ester derivatives thereof, as defined. Exemplary acylderivatives are the formyl, acetyl, propionyl, and succinyl derivatives,of which the formamide, aoetamide and succinyl derivatives arepreferred. Exemplary ester derivatives are methyl, ethyl and isopropylesters.

The mechanism underlying the present invention is believed to be thatthe methionine compound acts in vivo to reduce the effect of release bypolymorphonuclear leukocytes (PMNs) of hypochlorous acid and otheroxidants so that systemic oxidation especially of sulfhydrals,proteolysis, and tissue damage are inhibited. It is believed that the1-form of the methionine compound serves to fulfill its essential,recognized nutritional need that results especially in the presence ofserine or glycine to provide precursors for glutathione and otherantioxidants whereas it is the d-form that has a different action athigh dosage which is a well tolerated antioxidant and antiinflammatoryactivity.

To the extent that conditions benefiteed by the consumption ofdl-methionine are the result of a dietary deficiency of 1-methionine itmay be desirable to replenish methionine in food products as iscurrently done for a number of vitamins that are also made unavailableby food processing. The invention also employs methods and compositionsfor providing methionine in the final product for consumption in theamount that provides for replacement of unavailable methionine anadditional methionine that would accomplish the teachings herein whereit is desirable to obtain the additional antioxidant amount in a normalfood item. To safely provide low levels of additional dietary methionineit is necessary according to the invention to include additional glycineor serine in order to prevent the elevation of serum triglycerides whichoccur at relatively low doses of methionine.

Damico (U.S. Pat. No. 3,952,115) teaches the addition of N-acyl1-methionine as a preferred method to reduced undesired odor effects ofmethionine supplementation. He teaches that the amount of methionine tobe added to methionine-deficient protein can be determined by amino acidanalysis in the case of proteins known to below in methionine contentsuch that methionine should be added up to the level characteristic foregg protein (an amount recognized by the U.S. Food and Drugadministration as the upper limit for addition of methionine forcommercial foods). In the case of proteins for which methionine is lostby food processing such as extracted protein of soy bean he teaches thatthe amount of methionine to be added for proper nutrition involvesadding methionine derivatives as determined by rodent feedingexperiments.

Because of the role that inflammatory cells play in long term tissuedamage and because of the known dietary correlations of several seriousinflammatory pain conditions that may be affected by reduced control ofinflammatory cells, correction of a chronic marginal dietary deficiencyof methionine and thus improved long term control of inflammatory cellscan be expected to reduce the severity or incidence of these conditions.As an example of the possible contribution of reduced control ofinflammatory cells. especially PMNs, that may be due to a marginaldietary deficiency of available methionine, it had been shown thatproduces of stimulated PMNs can cause cellular transformationcharacteristic that has been associated with carcinogenesis (Weitzman,et. al., Science, 227:1231-3, 1985). Smokers that have been exposed toasbestos have very much higher lung cancer rates than exposednon-smokers, and it has been shown that smoking oxidizes a methionineresidue in alpha-1-protease inhibitor, thus allowing increased lungproteolysis. Other examples of damage due to reduced control of PMNsinclude arthritis and lung inflammation.

DESCRIPTION OF PREFERRED EMBODIMENTS

The best method to practice the teachings of described compounds dependson the particular conditions being treated and the compositions that arerequired to produce optimal results. plycine and serine are needed toprevent abnormal triglyceride levels which otherwise would occur whencompounds containing 1-methionine or its derivatives are consumed.

In addition, in those cases where deficiencies of other dietaryantioxidants may limit the total benefit to be derived from methioninecompounds they should be provided with the methionine compounds. Whenmethionine compounds are used in the upper portion of the dosage rangedissolution of the compound in the stomach should be slowed. Also,individuals that are more sensitive to gastric upset should be providedwith slow dissolving compositions to get effective relief.

For this purpose, the methionine compound and other antioxidants arepresent in an appropriate pharmaceutical dosage form, preferably in asustained release or controlled release form (e.g. an enteric coated orslow release form, which may be per se conventional), to enhance orensure release in the intestine rather than the stomach, optionally withsuitable excipients, such that each substance contributes its respectiveanti-inflammatory effect when a unit dosage of the composition isadministered. A preferred composition for human use is one where theunit dosage of the methionine compound, preferably dl- methionine, issufficient to provide a total daily dosage range of about 1.0 to 10gm/70 kg of body weight.

In another preferred aspect, the method employs a therapeuticantiinflammatory composition in unit dosage form comprising anantiinflammatory effective amount of at least on methionine compound asdefined above, and at least one member from the groups (a) through (e);(a) a triglyceride normalizing amount of glycine or serine as definedbelow, (b) at least one homocysteine affecting vitamin as defined above,in an amount sufficient to enable the systemic conversion, when consumedor administered, of homocysteine to methionine or cysteine, (c) at leastone dietary antioxidant in a synernistically antioxidant effectiveamount selected from a group of dietary antioxidants, (d) an inactiveexcipient that provides insolubility in the stomach and solubility inthe intestines; and (e) combinations thereof. Preferred dietaryantioxidants are vitamins A, C, E, selenium, or zinc, each preferably ina total daily dosage range of: vitamin A, 500 to 50,000 I.U.; vitamin C,1 to 1,000 mg; vitamin E, 1 to 150 I.U.; selenium, 1 to 200mcg; zinc, 1to 50 mg; and combinations thereof. Since the antioxidantcharacteristics, metabolism, and mechanism of methionine compoundsdiffer from those of other antioxidants, a synergistic antiinflammatoryeffect can be expected.

The benefit of glycine and serine especially, and vitamin B6 inference,for the adult male subject typically depends on their effect in reducingelevated triglycerides. Yanagita shows that in rats, 0.3% added1-methionine to the diet causes a more than three-fold increase in livertriglycerides (Yanagita, et. al., Agric. Biol. Chem, 48:815-6, 1984).Methionine and cysteine, the two amino acids which contain sulfur,constitute 3% of all the amino acid of proteins. Thus on a diet as lowas 10% protein the addition of 0.3% represents only a doubling ofdietary sulfur amino acids. Sugiyama, et. al., (Agric. Biol. Chem., 49:3455-61, 1985) showed that the addition of 2.5% glycine to a methioninesupplemented rat diet had no effect on plasma triglyceride levels. Thusthe triglyceride lowering effect in humans of glycine when added tomethionine is contrary to expectation. Betaine, while effective forlowering homocysteine was not effective for reducing triglycerides,other unexpected result. Elevated triglycerides are undesirable becausethey ar associated with increased incidence of gallstones (In:Epidemiology and Prevention of Gallstone Disease, L. Capocaccia, et al,eds., 1984, MTP Press, Ltd., Lancaster, p. 77-84). The benefit forreducing triglycerides is shown in the following human blood values:

    ______________________________________                                        Normal range for triglycerides                                                                        45-150  mg/dl                                         Consumption of dl-methionine, 1.5 gm/day                                                              224     mg/dl                                         (0.5 gm, 3 times per day) plus                                                1.0 gm/day betaine (0.5 gm 2 times/day)                                       Consumption of dl-methionine, 1.5 gm/day                                                              129     mg/dl                                         (0.5 gm, 3 times per day) plus                                                1.5 gm/day glycine (0.5 gms 3 times/day)                                      ______________________________________                                    

These results show that glycine but not betaine was effective inallowing normal blood values for triglycerides during daily consumptionof methionine by a human subject.

In another preferred aspect, the invention concerns a method fortreating thiol depletion in a subject. The method comprisesadministering to a subject a composition in dosage form containing aneffective thiol raising amount of at least one methionine compound withglycine or serine. Preferably for this purpose, the methionine isadministered in a total daily dosage range of about 1 to 10 gm/70 kg ofbody weight and is continued daily until the thiol levels arenormalized. In a preferred embodiment, the dl-form of methionine is usedwith glycine and serine, preferably in a daily oral dose of 2.0 gramstaken 2 times in spaced equal doses with 1.0 gram of glycine and serinetaken at 2 spaced equal doses. In another preferred embodiment seleniumis taken with the methionine and glycine or serine in a total daily doseof about 2 to 200 micrograms.

In another preferred aspect, the invention concerns a therapeuticantiinflammatory composition in unit dosage form comprising anantiinflammatory effective amount of at least one methionine compound asdefined above, and a triglyceride normalizing amount of glycine orserine as defined above in Lipid Normalization.

In another preferred aspect, the method employs a therapeuticcomposition in unit dosage form for treating thiol depletion in asubject comprising a thiol increasing amount of the 1-form of methionineand a thiol sparing amount of the d-form of methionine, as definedabove, and a triglyceride normalizing amount of glycine or serine asdefined below, and at least one member from the groups (a) and (b); (a)at least one homocysteine affecting vitamin as defined above, in anamount sufficient to enable the systemic conversion, when consumed oradministered, of homocysteine to methionine or cysteine, (b) an inactiveexcipient that provides insolubility in the stomach and solubility inthe intestines: and (c) combinations thereof.

An example of the thiol repletion benefit of dl-methionine and glycineis shown for a HIV-positive subject with AIDS associated diseaseconditions. The patient had increased sedimentation rate for red bloodcells, leukoplakia (monilia) and had been losing weight over a 7 monthperiod prior to treatment with dl-methionine, 1 gram twice a day,glycine, 0.5 gm twice a day, folate, 1 mg per day and one multivitamin.After 2 months treatment weight loss had been arrested (gained 3 lbs.),the leukoplakia (thrush) had resolved and the red cell sedimentationrate dropped to 41 from a high of 105. Based on the antioxidant andmetabolic properties of dl-methionine and on the reduced blood levels ofglutathione in symptom-free HIV-positive subjects the methionine-glycinebenefit seen in the example above is thought to be moderated by thiolrepletion.

Detailed methods for the preparation and tableting of methioninecompounds are contained in U.S. Pat Nos. 4,902,718, 4,927,850,5,053,429, and 5,084,482, which methods are incorporated herein byreference.

Having thus described our invention, what we claim and desire by LettersPatent to secure are the following:
 1. A method for ameliorating theinflammatory symptoms of human immunodeficiency virus infection and AIDSin man employing the administration to the subject in need thereof atherapeutic composition in dosage form consisting essentially of ananti-inflammatory amount of methionine compound selected from the groupconsisting of dl-methionine, methyl, ethyl, propyl and isopropyl estersthereof, N-acetyl-dl-methionine, N-formyl dl-methionine and methyl,ethyl, propyl and isopropyl esters of N-acetyl and N-formlydl-methionine to provide a total daily methionine dosage in the range of1.0 to 10 grams per 70 kg of body weight and further comprising at leastone triglyceride normalizing compound selected from the group consistingof serine and glycine, the serine and glycine are present from 1/5 to 3times the methionine compound present.